Drug-induced epigenetic remodeling to prevent fibrosis

ABSTRACT

A therapeutic treatment for preventing or reducing the formation of fibrosis comprising administering to a patient a UTX or JMJD3 inhibitor that are effective in preventing or reducing fibrosis in situations wherein access to an injury or dysmorphogenetic tissues before the fibrotic process becomes established in the tissues.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of International Patent ApplicationNo. PCT/US17/53249, filed Sep. 25, 2017, published as WO 2018/058034 onMar. 29, 2018, which claims benefit to U.S. Provisional Application Ser.No. 62/398,926, filed Sep. 23, 2016, the disclosure content of which ishereby incorporated by reference in its entirety.

FIELD OF INVENTION

The present invention is generally related to blocking histone modifyingactivity of H3K27 demethylases, such as Ubiquitously Transcribed XChromosome Tetratricopeptide Repeat Protein (UTX), which would allow forfaster accumulation of H3K27me3 and therefore a condensed structure onchromatin on the newly replicated DNA that could prevent myofibroblastdifferentiation by blocking association of the master lineage-specifyingtranscription factor MRTFA.

BACKGROUND OF INVENTION

Most tissues of the eye are susceptible to developing fibrotic disease,blinding millions of people throughout the world. Furthermore, fibrosiscan affect almost every organ of the body, thus creating a diseaseprofile that affects tens of millions of people worldwide. Despite thefar reaching effects of fibrosis, there are no effective approaches toprevent, slow or reverse this disease process. The cell type responsiblefor causing fibrotic disease is the myofibroblast. Understanding how acell acquires an altered heritable phenotype to become a myofibroblast,leading to fibrotic scarring associated with this pathological diseaseprocess, is a key question, likely to provide essential clues towarddeveloping anti-fibrotic therapeutics, which remains unanswered.

Changing transcriptional programming during reprogramming of a cell to amyofibroblast is not well understood. In major aspects, it has to relyon changes in the epigenetic mechanisms of inheritance of chromatinstructure during DNA replication. The mechanism of epigeneticinheritance during cell proliferation remains unknown, and we know evenless about how epigenetic information and the correspondingtranscriptional programs change during cell reprogramming. The gaps inour knowledge of these essential biological processes are based on thelack of direct experimental approaches that would allow examining thestructure of chromatin and the state of transcription during andfollowing DNA replication during cell proliferation and celldifferentiation.

How cells with a normal function are reprogrammed to becomemyofibroblasts, the cell type responsible for fibrotic disease, is notyet understood. Fundamental to the regulation of this celldifferentiation process is uncovering how chromatin structure controlsaccess of specific transcription factor (TFs) to DNA to coordinatechanges in transcriptional programs and thus cell lineage specification.

SUMMARY OF INVENTION

De-condensed structure of nascent, post-replicative chromatin isessential for binding of lineage-specific transcription factors torepressed genes and changing of the transcriptional programs ofdifferentiating cells. Blocking histone-modifying activity of specificenzymes has the potential to manipulate the structure of nascentchromatin. Therefore we determined that blocking the histone modifyingactivity of histone H3K27 trimethylase (KDM) UTX leads to a fasteraccumulation of H3K27me3 and therefore a much more condensed nucleosomestructure on nascent DNA, and prevents binding of masterlineage-specifying transcription factor MRTF-A and myofibroblastdifferentiation. Inhibition of UTX activity effectively prevented theemergence of αSMA+ myofibroblasts. These findings provide insight intothe epigenetic events regulating cell reprogramming to a myofibroblastphenotype as well as reveal the potential to develop therapeuticstrategies to modulate epigenetically-mediated cell reprogramming totreat fibrotic disease.

A method of preventing or reducing fibrosis, comprising administering toa patient a pharmaceutical composition that inhibits UTX H3K27de-methylase activity, which would lead to closed chromatin structure onnascent DNSA, and block reprogramming to a myofibroblast phenotype, themajor cell type associated with causing fibrotic disease.

The method of claim 1 wherein the H3K27 de-methylase UTX inhibitor isGSK-J4.

A method for reducing fibrosis formation comprising treatment with aH3K27 de-methylase UTX inhibitor, wherein said treatment prevented 1) asmooth muscle actin (αSMA) expression, a defining feature ofmyofibroblasts, in the ex vivo mock cataract surgery model and 2)expression of Fibronectin EDA (FN-EDA) and Collagen I expression,wherein said FN-EDA expression is tightly linked to myofibroblastdifferentiation and Collagen I is a defining feature offibrosis/scarring.

A method of treatment of tissues before the fibrotic process establishescomprising administering to a patient treatment with a H3K27de-methylase UTX inhibitor.

A method of treatment wherein treatment with H3K27 de-methylase UTXinhibitor can be applied at the time of cataract surgery to prevent thedevelopment of the lens fibrotic disease Posterior Capsule Opacification(PCO).

A method of preventing or reducing fibrosis, comprising administering toa patient an effective amount of a pharmaceutical composition comprisinga H3K27 de-methylase UTX inhibitor.

A method of preventing or reducing fibrosis, comprising administering toa patient an effective amount of a pharmaceutical composition comprisinga H3K27 de-methylase UTX inhibitor, wherein the H3K27 de-methylase UTXinhibitor is GSK-J4 or a suitable salt thereof.

A method of preventing or reducing fibrosis, comprising administering toa patient an effective amount of a pharmaceutical composition comprisinga H3K27 de-methylase UTX inhibitor, wherein the H3K27 de-methylase UTXinhibitor is GSK-J4 or a suitable salt thereof, wherein thepharmaceutical composition is administered topically to a skin surface.

A method of preventing or reducing fibrosis, comprising administering toa patient an effective amount of a pharmaceutical composition comprisinga H3K27 de-methylase UTX inhibitor, wherein the H3K27 de-methylase UTXinhibitor is GSK-J4 or a suitable salt thereof, wherein thepharmaceutical composition is administered topically to a skin surface,wherein the pharmaceutical composition is administered to a patientwithin 24 hours of a surgical procedure.

A method of treatment of a patient tissues before the fibrotic processestablishes after a surgical procedure, comprising administering to saidpatient an effective amount of a pharmaceutical composition comprising aH3K27 de-methylase UTX inhibitor at the time of the surgical procedure.Preferably, wherein the H3K27 de-methylase UTX inhibitor is GSK-J4 or asuitable salt thereof. The route of administration may be selected bythose of skill in the art based upon the injury to be treated,preferably, wherein the pharmaceutical composition is administeredorally, intradermally, or topically to a skin surface.

A method of preventing or reducing the formation of fibrosis in oculartissues after an ocular surgery comprising: administering to a patientundergoing said ocular surgery a UTX inhibitor at the time of the ocularsurgery. Preferably, wherein the UTX inhibitor is GSK-J4 or a suitablesalt thereof. Preferably, wherein the UTX inhibitor is administeredtopically to the eye.

A method of treatment of the fibrotic disease Posterior CapsuleOpacification (PCO) comprising: performing a cataract surgery on apatient; and administering to said patient a sufficient amount of a UTXinhibitor during said surgery, preferably, wherein the UTX inhibitor isGSK-J4 or a suitable salt thereof, and preferably, wherein the UTXinhibitor is administered topically to the eye.

A method of treatment of the fibrotic disease Posterior CapsuleOpacification (PCO) comprising: performing a cataract surgery on apatient; administering to said patient a sufficient amount of a UTXinhibitor during said surgery; and implanting an intraocular lens intothe eye, wherein said intraocular lens comprises a sustained releasemechanism to allow for release of the UTX inhibitor for at least 24hours. In certain embodiments, said sustained release mechanism providesfor release for between 24 and 72 hours.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a model depicting how chromatin structure can bemanipulated using the UTX/JMJD3 inhibitor GSK J4 to block reprogrammingto a myofibroblast phenotype leading to fibrosis.

FIGS. 2A-2C illustrate ex vivo mock cataract surgery woundrepair/fibrosis model and the process of wound healing, which iscompleted by day 3, as shown in 2C.

FIGS. 3A and 3B illustrate cells migrating from ex vivo explant onto therigid tissue culture substrate called the ECZ provides an ideal model tostudy mechanism of myofibroblast emergence and shows that CD44+ leadercells that serve as the myofibroblast progenitors are highlyproliferative

FIG. 4 shows that αSMA positive myofibroblasts emerge at the leadingedge of the ECZ by day 3.

FIG. 5 illustrates a schematic diagram of the Chromatin Assembly Assay(CAA), which reveals how chromatin structure is re-established followingDNA replication.

FIG. 6 shows the period of period of ‘open’ post-explicative chromatinin the ex vivo mock cataract surgery cultures, revealing the time periodof cell reprogramming to a myofibroblast phenotype.

FIGS. 7A-7C illustrate a diagram of the H3K27 modifying enzymes,immunolocalization studies for EZH2 and H3K27me3 in the ECZ, and CAAshowing that EZH2 accumulation on day 2 in the ECZ.

FIG. 8 illustrates that UTX expression is increased in response toinjury in the ex vivo mock cataract surgery cultures.

FIGS. 9A-9C illustrate how the pro-fibrotic transcription factor MRTF-Ais activated and 9B depicts that MRTF-A recruitment to DNA occurs duringDNA replication, while 9C depicts a thymidine block.

FIGS. 10A-10B show that treatment with the UTX inhibitor GSK J4 preventsa smooth muscle actin (αSMA) expression in the ex vivo mock cataractmodel.

FIG. 11 shows that treatment of human lens cells (FHL 124 cell line)with GSK J4 suppresses the expression of Fibronectin EDA (FN EDA) andCollagen I, ECM molecules associated with fibrosis.

FIGS. 12A and 12B show that treatment with GSK J4 slows but does notblock wound healing in the ex vivo mock cataract surgery explants over athree day period.

FIG. 13 shows that short-term treatment of the ex vivo mock cataractsurgery explants with GSK J4 can effectively suppress Fn-EDA and αSMAexpression associated with fibrosis.

FIGS. 14A-14C show that short-term treatment with GSK J4 can effetelysuppress myofibroblast emergence without blocking wound healing in theex vivo mock cataract surgery explants.

FIG. 15 illustrates changes in post-replicative chromatin that allowsbinding of pro-fibrotic TFs during reprogramming to a myofibroblast.

FIG. 16 illustrates how using epigenetic inhibitors to UTX/JMJD3 canmanipulate chromatin structure to block reprogramming to a myofibroblastphenotype.

FIG. 17 depicts line structures of certain UTX/JMJD3 inhibitorsincluding: GSK-J1, GSK-J2, GSK-J4, and GSK-J5.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Fibrotic disease affects almost every organ of the body and is believedto result in approximately 45% of all deaths in the industrializedworld. This staggering number is underscored by the fact that we have noreadily available treatments or therapeutics to treat fibrosis. Whileall tissues can become fibrotic, the tissues of the eye are susceptibleto developing fibrosis, blinding millions of people throughout theworld. This disease process, regardless of the tissue type affected, ischaracterized by excessive extracellular matrix (ECM) production leadingto scarring and loss of tissue function. There are no effectiveapproaches to prevent, slow or reverse this disease process. The celltype responsible for causing fibrotic disease is the myofibroblast,which can result from the reprogramming of many distinct cell typesincluding fibroblasts, pericytes, and epithelial cells that haveundergone a mesenchymal transition (EMT). Understanding how these cellsacquire an altered phenotype to become a myofibroblast responsible forscarring associated with fibrotic disease is key to developinganti-fibrotic therapeutics.

Cell reprogramming to a myofibroblast phenotype is triggered by specifictranscription factors (TFs) that induce changes in cell phenotype.However, we do not understand how these reprogramming changes areinduced at the molecular level. Lineage-specific TFs overcome thebarrier of condensed arrays of nucleosomes that are associated withrepressed genes which have to be activated during cell reprogramming.Therefore, reprogramming events must involve changes in epigeneticmarkings of chromatin, such as covalent modifications of histones byhistone modifying enzymes, which results in alteration of chromatinstructure and thereby facilitate binding of TFs and impact genetranscriptional programs. These issues are of general biologicalsignificance to fibrotic diseases throughout the body, as they mergeseveral key phenomena, transcription, epigenetics, chromatin structureand cell reprogramming.

While there is data to suggest that the fibrotic process is underepigenetic control, we are only in the infancy of understanding thisemerging field. Studies on this topic so far show a role for DNAmethylation in regulating fibrosis, with little understanding to dateabout the role of histone modifications. Herein, we describe mechanismsof action and therapeutic treatments and methods for modifyingepigenetic and transcriptional mechanisms that are utilized during veryearly stages of reprogramming for induction of fibrotic disease.Accordingly, for any number of fibrotic diseases, we teach therapeuticsand methods that can be utilized to alter the process of differentiationof myofibroblasts to prevent or reduce the formation of fibrosis.

As known to those of ordinary skill in the art, DNA is wrapped aroundnucleosomes (chromatin structures composed of several histone proteins)which allow DNA to maintain its compact structure and regulate geneactivity. The maintenance of nucleosome arrays, and therefore chromatinstructure, is provided by so-called epigenetic mechanisms duringdifferent stages of the cell cycle. Cell differentiation also relies onepigenetic mechanisms, although the exact way that changing chromatinstructure and gene activity impacts differentiation is poorlyunderstood. The phenotypic outcome of cell reprogramming to amyofibroblast, the major cell type associated with fibrosis and fibroticdisease progression is altered by targeting epigenetic mechanisms.

Herein, we show that reprogramming of cells normally tasked with repairto a myofibroblast phenotype requires transient de-condensation ofchromatin/nucleosome structure during early stages of DNA replication.This “opening” of the chromatin exposes regulatory sequences in the DNAof repressed genes and facilitates binding of lineage-specifyingtranscription factors (TFs) to DNA. Tri-methylated lysine 27 of Histone3 (H3K27me3) is a key epigenetic mark associated with the most condensed(‘closed’) chromatin structure. Importantly, H3K27me3 marks allrepressed genes in the genome, among which are genes that have to beactivated to change transcriptional programs during celldifferentiation.

Histone modifying enzymes for lysine 27 of histone H3 (H3K27) includetwo groups of enzymes with antagonistic activities: 1) H3K27methyltransferases (HMT) EZH1 and EZH2 that catalyze the addition ofmethyl groups to H3K27, and 2) H3K27me3 de-methylases (KDM) UTX andJMJD3, involved in catalyzing the removal of methyl groups fromH3K27me3. Thus, targeting of KDM enzymes with small molecule inhibitorsleads to an increase of H3K27me3, and as a result to a more closed,condensed structure of chromatin (rapid accumulation of H3K27me3) onnewly replicated DNA. This condensed structure of chromatin blocksbinding of TFs essential for reprograming to myofibroblasts and willprevent or slow fibrosis.

Therefore, the presence of H3K27me3 in the genome strongly correlateswith the condensed structure of nucleosomes. Accordingly, H3K27me3provides for a reliable readout of chromatin compaction. We have shownthat inhibitors of enzymes responsible for de-methylating of H3K27,which allow for faster accumulation of H3K27me3 and a closed chromatinstructure would block the ability of pro-fibrotic TFs to bind to the DNAand the development of fibrosis.

Accordingly, herein, we describe therapeutic treatments for preventingor reducing the formation of fibrosis. These treatments include use ofparticular H3K27me3-specific KDMs blocking agents, and methods of use ofthe same in procedures to prevent the formation of, or reduce theformation of fibrosis. Accordingly, broadly, the embodiments of thepresent disclosure relate to compositions and methods to generateeffective epigenetic-based therapeutic approaches to slow or preventfibrotic disease. Further embodiments are directed towards compositionsand methods to prevent the myofibroblast phenotype expression withfibrosis-promoting extracellular matrix proteins and fibrotic diseaseprogression.

The therapeutic treatments described herein are effective in preventingfibrosis in situations in which we can access an injury ordysmorphogenic tissue before the fibrotic process becomes established inthe tissue. Fibrosis is a widespread issue, whether related to theexcess formation of scar tissues, which has created an entire line ofmedicine related to plastic surgery—or reduction of fibrosis formation.

Indeed, fibrosis formation is problematic in pulmonary fibrosis, cysticfibrosis, cirrhosis, atrial fibrosis, endomyocardial fibrosis, glialscarring, arterial stiffening, arthrofibrosis, crohn's disease, keloids,myelofibrosis, systemic fibrosis, etc. Fibrosis simply covers everyorgan and skin type of the body. Many of these diseases are slowprogressing, and treatment may be suitable herein, to reduce theformation of fibrosis or slow disease progression.

However, certain acute fibrotic events are also of interest, forexample, scarring due to surgical procedures. Formation of fibrosis inthese instances can be reduced or eliminated, and thus prevent suchtissue injury or, for many, the mental components that go with theformation of fibrotic tissue. One area that may be targeted areinjury/surgery/disease in the visual system including the lenspost-cataract surgery, fibrotic disease, Posterior Capsule Opacification(PCO), hazing/opacification of the cornea following corneainjury/surgery including Lasik, age-related macular degeneration (AMD),scarring following surgery of all types, scarring following skin injury.

For example, in a PCO model, using an ex vivo lens mock cataract surgerymodel in chick embryos that recapitulates features of PCO, weinvestigated the cell reprogramming events involved in regulatingchanges in epigenetic markings of chromatin, which result in alterationsof chromatin structure and impact gene transcriptional programs tocontrol the emergence of myofibroblasts. Before surgery and until thefirst day following induction of cell reprogramming, we found thatchromatin is characterized by a significant delay in the accumulation ofthe key repressive histone mark H3K27me3 on the myofibroblastprogenitors following DNA replication. This signifies an “open’ nascentchromatin structure, revealing a potential window of opportunity for therecruitment of pro-fibrotic TFs, such as MRTF-A, to DNA that arerequired for the reprogramming of the progenitor cells to amyofibroblast.

Following this H3K27me3 rapidly accumulated on nascent DNA afterreplication, consistent with ‘closed’ nascent chromatin, reflective of atight structure of chromatin that would prevent association of unwantedTFs to DNA to maintain the newly acquired differentiated fibroticphenotype. Changes in the rate of accumulation of H3K27me3 methylationassociated with the development of fibrosis can be regulated by H3K27modifying enzymes, which include the H3K27 tri-methylase EZH2 and H3K27demethylase UTX.

Using newly developed experimental paradigms as described herein, wefound that epigenetic marking during cell proliferation relies not onthe transfer of modified histones to daughter strands, but rather onstable association of multiple histone-modifying proteins during DNAreplication. Similar results were obtained in multiple lens models ofcell reprogramming leading to fibrotic disease. Lens cells before injuryand until the first day following surgery in the ex vivo chick modelhave chromatin that is characterized by a significant delay in theaccumulation of the key repressive histone mark H3K27me3 following DNAreplication. This signifies a de-condensed structure of nucleosomes.

The same ‘open’ post-replicative chromatin was also discovered in mouseand human lens cells during their induction to the myofibroblastphenotype, suggesting that this is a previously unknown pivotal propertyof all myofibroblast progenitor cells. Accordingly, our data not onlysupports the therapeutic treatments in animal models, but translatesthrough to human cells. Our data confirms that this ‘open’ state ofpost-replicative chromatin is more amenable to binding of newly inducedspecific transcription factors (TFs) essential for cell reprogramming.Importantly, the state of ‘open’ chromatin may be manipulated in orderto change the ability of TFs to associate with their target sites on DNAto therapeutically target myofibroblast differentiation.

Fibrosis, while impacting nearly every tissue in the body, has aparticular impact on and affects most tissues of the eye, leading toblindness in millions of people worldwide. Few options exist to treatthe scarring associated with this pathological disease process. The keycellular mediator of fibrosis is the myofibroblast characterized by itsα-smooth muscle actin (αSMA)-mediated contractile function and synthesisof an altered extracellular matrix environment.

We focused on understanding how a cell type is reprogrammed to acquire anew transcriptional profile that induces the cell to become a fibroticdisease-causing myofibroblast in order to develop therapeutic tools totreat fibrotic disease. While studies to date have focused on the growthfactors and receptors that signal the transition to a myofibroblastphenotype, few studies have addressed the epigenetic mechanisms thatunderlie this phenotypic change. Epigenetic regulation is thought to beessential to the differentiation of other cell types, including stemcells. While we know the end result of epigenetic changes during celldifferentiation, the mechanistic aspects of epigenetic inheritance thatchange a cell's phenotype during differentiation still remain a mystery.This lack of knowledge is mostly due to the absence of experimentalapproaches for investigating chromatin structure during DNA replicationthe disruptive cell cycle stage during which chromatin is remodeled.

We developed methods that allow, for the first time, examination of thenature of epigenetic marking of chromatin and the re-assembly ofproteins on DNA during replication. Herein, we describe experimentalapproaches regarding epigenetic mechanisms regulating myofibroblastdifferentiation and the preservation of the myofibroblast phenotypewhich results in tissue scarring. For these studies we used an ex vivomock cataract surgery model of the lens fibrotic disease, PosteriorCapsule Opacification (PCO), as well as human lens culture models inwhich epithelial mesenchymal transition (EMT) leading to fibrosis isinduced by exposure to TGFβ. Using these model systems we studied theepigenetic cell reprogramming to a fibrotic disease causingmyofibroblast on a single cell level, as well as tested anti-fibrotictherapeutics. Because of the conservation of myofibroblastdifferentiation, therapeutic strategies described herein will generate aconsistent effect for modifying myofibroblast differentiation regardlessof the cellular source or tissue type, therefore our findings have wideapplication for the treatment of fibrosis in many tissues.

Methods and Examples

FIG. 1 describes the manipulation of chromatin structure usingepigenetic inhibitors to block pro-fibrotic transcription factors fromassociating with DNA to prevent the onset and progression of fibroticdiseases. FIG. 1 (top) depicts an “open” chromatin structure, whereinthe structure is able to be bound by transcription factors, which can,in some cases re-program cells to a myofibroblast fate leading tofibrotic disease. Instead, by blocking the enzymatic activities of theH3K27me3 de-methylases UTX/JMJD3 with a therapeutic, in this caseGSK-J4, the structure of the chromatin becomes ‘closed’ and preventsbinding of pro-fibrotic transcriptions factors, such as MRTF-A.Subsequently, cell reprogramming to a myofibroblast phenotype andchanges in the ECM environment associated with onset and progression offibrotic disease are prevented.

We tested this epigenetic targeting strategy using a clinically relevantex vivo chick mock cataract surgery model that recapitulates the majorfeatures of the lens fibrotic disease, PCO, as well as a human lenscells (SRA01/04). Human SRA01/04 lens cells are studied extensively forthe acquisition of a mesenchymal phenotype resulting from an EpithelialMesenchymal Transition (EMT) after exposure to TGFβ associated with thedevelopment of lens fibrosis [4, 5]. In studies with the SRA01/04 humancell line we now show that TGFβ induces expression of FN-EDA andcollagen I, both components of a fibrotic-promoting ECM environment.Importantly, we found that the epigenetic paradigm for myofibroblastemergence that we discovered in our studies with the ex vivo mockcataract surgery cultures is replicated in these lens cell upontreatment with TGFβ.

FIG. 2A illustrates how the ex vivo mock cataract surgery explants arecreated. Ex vivo mock cataract surgery explants provide a powerful modelin which to study wound healing as well as the development of fibroticdisease. Briefly, to create this model, the fiber cell mass ishydroeluted from the lens leaving behind lens epithelial cells thatremain adherent to the lens capsule. The resultant ex vivo explant whenplaced in culture is star shaped, with the adherent cells located in thepoints of the star. In response to the injury induced by the surgery,mesenchymal cells involved in repair move to the wound edges.

As shown in FIG. 2B wound healing across the denuded basement membraneis typically completed by day 3. Lens epithelial cells led by a leadercell population, which expresses the surface receptor CD44+[42], movecollectively to repopulate the denuded area of the wounded lens capsule(where the differentiated lens fiber cells were removed). FIG. 2Cdepicts the wound area over a three-day period. In FIGS. 3A and B CD44+leader cell population that has migrated to the outside wound edge ofthe explants (FIG. 3A, left panel, right arrow) directs the collectivemigration of lens epithelial cells onto the rigid tissue culturesubstrate, a region we refer to as the extracapsular zone (ECZ). FIG. 3Bshows the CD44-rich highly proliferative population at the leading edgeof lens epithelial cells migrating across the tissue culture plastic.FIG. 4 demonstrates that αSMA+ myofibroblasts first appear at theleading edge of the ECZ on day 3, preceded by the assembly of afibronectin EDA (FN-EDA) matrix environment at day 2, an ECM shown to bea key fibrosis-inducing factor. Therefore, within one model system wecan assess epigenetic targeting strategies for preventing fibrosis anddetermine how these approaches will affect normal wound healing. This exvivo model was used to investigate the epigenetic mechanisms (detailedbelow) involved in regulating cell reprogramming to a myofibroblastphenotype associated with lens fibrotic disease.

To study the epigenetic mechanisms associated with cell reprogramming toa myofibroblast phenotype we are using a state of the art assay calledthe Chromatin Assembly Assay (CAA). FIG. 5 is a schematic diagram of theCAA used to reveal how chromatin structure is re-established followingDNA replication. Briefly, DNA is labeled in vivo in a pulse-chase mannerwith EdU, which is then chemically conjugated with biotin using a‘Click-iT’ reaction. The proximity of a protein of interest to nascentDNA is then examined by Proximity Ligation Assay (PLA, Olink,Bioscience). In PLA, both chromatin-associated protein and biotinlabeled DNA are detected with primary antibodies and then with secondaryantibodies that are conjugated with short strands of DNA. Addition ofcomplementary linker oligonucleotides allows formation of a DNA circleif antibodies are within approximately 40 nm of each other. DNA circlesare then amplified by rolling circle amplification (RCA), fluorescentlylabeled oligonucleotides are added that will hybridize to the RCAproduct to generate a fluorescent signal. PLA is a powerful single celltechnique that detects single molecule interactions with highsensitivity (10⁻⁴⁰ M) and specificity. Cells can be then immunostainedwith any additional antibody to examine the specificity of interactions(e.g. specific PLA signals are detected only in EdU-labeled cells), andto assess tissue specificity using different protein markers. Reliablepulse-chase EdU labeling in cells can be detected from 3-5 min toseveral hours. CAA is a rapid and convenient method to examine thekinetics of assembly of proteins and histones on daughter DNA afterreplication at a single cell level. PLA appears as punctuate dots withinEdU labeled nuclei. The example shows accumulation of H3K27me3, a markof closed chromatin, accumulating on nascent DNA. In the example shownin FIG. 5 (bottom) the chromatin is closed.

FIG. 6 defines the period of ‘open’ post-explicative chromatin in the exvivo mock cataract surgery model using CAA. Using the ex vivo model wefirst wanted to determine when nascent chromatin is ‘open’ to indicatethe time period in which cell reprogramming to a myofibroblast may beoccurring. The results indicate that the induction of myofibroblastdifferentiation is accompanied by a several hours delay in accumulationof H3K27me3 to nascent DNA. Thus, indicating a period of time betweenday 1 and day 2, when cell reprogramming to a myofibroblast isoccurring. Lens epithelial cells prior to injury and cells in the ECZregion 1 and 2 days after surgery were labeled with EdU for 30 min,followed by 2 hour chase. CAA was performed for H3K27me3, followed byimmunostaining for biotin (EdU). PLA signals only (dots) are shown inlower panels. In contrast, on day 2 after injury, H3K27me3 accumulatesrapidly, indicating that by day 2 nascent chromatin is closed.

FIG. 7 illustrates the major H3K27 modifying histone enzymes: Histonemethyltransferase EZH2, which adds methyl groups to H3K27 and thehistone de-methylase UTX, which removes methyl groups from H3K27.Manipulating activity of these enzymes is a major way to modify H3K27,and to change chromatin structure. FIG. 7B shows co-immunostaining ofH3K27me3 and the H3K27 tri-methyltransferase EZH2 in lens cells from theECZ region of the ex vivo cataract surgery explant model before andafter injury. EZH2 expression increased in leader cells coincident withthe increase in H3K27me3 post injury. FIG. 7C shows the association ofEZH2 on labeled nascent DNA in the ECZ region of the lens 1 day and 2days after mock cataract surgery. CAA was performed for EZH2 and forbiotin (EdU). PLA (dots) are shown in lower panel. On day 2 EZH2 rapidlyaccumulates on nascent DNA coincident when we observed the accumulationof H3K27me3. Thus, we have defined an open period of chromatin (absenceof H3K27me3 and EZH2 accumulation) on day 1 to day 2, when reprogrammingto a myofibroblast can occur. By day 2, we have established thatchromatin is closed, and that no new transcriptional program canpotentially be acquired.

FIG. 8 shows that expression of the H3K27 de-methylase UTX is increasedin response to injury. Therefore, the H3K27 demethylase, UTX is poisedto prevent methylation of H3K27 on day 1, to keep chromatin in the openstate. Thus, manipulation of demethylase activity represents a keyepigenetic strategy to induce closure of chromatin to prevent theadoption of new transcription program associated with the onset offibrotic disease. We tested this below in FIGS. 10-13.

FIG. 9A depicts a model of activation of MRTF-A downstream of the TGFβand ECM mediated mechanical signaling, leading to induction ofpro-fibrotic genes. FIG. 9B shows MRTF-A recruitment to nascent DNAoccurring only during DNA replication. One day after surgery, cells ofthe ECZ region were labeled with EdU for 30 min, followed by 60 minchase. Cells were then incubated with thymidine for 24 hr. Thymidine wasremoved and cells were kept for additional 2 and 4 hr. CAA was performedfor MRTF-A followed by immunostaining for biotin (EdU). The upper panelin FIG. 9B shows staining with DAPI, EdU and PLA; the lower panel showsPLA dots only. These findings indicate that recruitment of thepro-fibrotic lineage transcription factor MRTF-A occurs only during DNAreplication. FIG. 9C depicts a chart of DNA synthesis and impacts ofthymidine block.

Next, we tested if manipulating histone de-methylase activity couldchange chromatin structure to block pro-fibrotic transcription factorsfrom binding to DNA and the acquisition to a myofibroblast phenotypeassociated with fibrosis. FIG. 10A shows that the selective JMJD3/UTXinhibitor, GSKJ4 effectively suppressed the emergence of myofibroblastsassociated with the development of fibrotic disease. FIG. 10 A, B showthat treatment with GSKJ4 prevents αSMA expression in cells from the exvivo mock cataract surgery explants. Thus, treatment with GSKJ4 preventsαSMA expression in the ex vivo mock cataract surgery model.

We also performed similar studies with human lens cell culture. FHL124human lens cells, a non-transformed spontaneously generated human lenscell line from embryonic explants, were stimulated with TransformingGrowth Factor β (TGFβ), a potent inducer of the fibrotic response, inthe absence or presence of the UTX inhibitor GSKJ4. Western blotting wasperformed with antibodies to extracellular matrix molecules associatedwith fibrosis, including Fibronectin EDA (FN-EDA), a splice form of FNand Collagen I. FN EDA expression is tightly linked to myofibroblastdifferentiation and Collagen I is a defining feature offibrosis/scarring. GAPDH was used as a loading control for WB analysis.FIG. 11 shows that treatment with the UTX inhibitor, GSKJ4 blocked TGFβinduced expression of both FN EDA and Collagen I, effectively preventingchanges in ECM associated with the onset and progression of fibrosis.

Collectively, FIGS. 10 and 11 indicate that blocking enzymatic activityof the H3K27 de-methylase UTX, leads to closed chromatin structure, andthus blocked reprogramming to a fibrotic phenotype. Accordingly,therapeutics that block the enzymatic activity of H3K27 de-methylase UTXcan prevent fibrosis formation, by closing the chromatin structure andprevent the reprogramming of the myofibroblasts to a fibrotic phenotype.

We also determined whether GSKJ4 treatment affected wound healing. It isadvantageous to identify an anti-fibrotic therapeutic with limited to noeffect on the normal wound healing process. FIG. 12 shows that woundhealing is not prevented by treatment with the JMJD3/UTX inhibitorGSKJ4. The Ex vivo mock cataract surgery explants exposed to GSKJ4somewhat slowed but did not prevent wound healing. Open wound area (μm²)was measured on Day 0 (D0) through Day 3 (D3) in the presence of vehiclecontrol (DMSO) or GSKJ4. Open wound area was measured for each ex vivocapsule D0-D3 and presented in the graph+/−SEM. Wound healing wasslightly delayed by GSKJ4 treatment. Representative phase images of exvivo mock cataract surgery explants are shown on D0-D3.

Next, we tested whether a short-term treatment of GSKJ4 could beeffective to block fibrosis without any effect on wound healing. FIG. 13shows that short-term treatment with GSKJ4 was effective in suppressingexpression of Fn-EDA and αSMA associated with fibrosis. Cells from theex vivo mock cataract surgery explants were treated with vehicle orGSKJ4 for 24 hr. ECZ regions were treated 1 day after injury andinhibitor was washed away after 24 hour of treatment. We chose to addthe inhibitor on day 1, during the time of open chromatin, the perfecttiming to prevent cell reprogramming to a fibrotic phenotype. Lysateswere collected on day 3, the time when αSMA is typically expressed, andprocessed for western blot analysis for expression of Fn-EDA, αSMA andGAPDH (loading control). Short-term treatment with GSK-J4 significantlyinhibited αSMA and Fn-EDA expression indicating that a shorter pulse oftreatment is effective for treating fibrosis.

FIGS. 14A and B shows that short-term treatment with GSK-J4 waseffective to block myofibroblast emergence without preventing woundhealing from occurring. Cells from the ex vivo mock cataract surgeryexplant were treated for 24 hour as indicated above with vehicle orGSK-J4. Lysates were collected on day 3 and examined by western blotanalysis for αSMA and GAPDH expression. The graph represents the ratioof αSMA to GAPDH+/−SEM. Treatment with the JMJD3/UTX inhibitoreffectively blocked αSMA expression. In FIG. 13B we determined theeffect of GSKJ4 short-term treatment on wound healing. For thesestudies, ex vivo cultures were treated for at the time of injury, day 0,with vehicle or GSK-J4 for a 24 hour period. Wound healing was thenfollowed from day 0 through day 4. Phase images shown and the graphindicates open wound area +/−SEM. These data confirm that short-termtreatment with GSKJ4 does not affect wound healing in response to injuryin the ex vivo mock cataract surgery explants.

FIGS. 13 and 14 indicate that blocking enzymatic activity of the H3K27demethylases with short-term treatment of GSKJ4 can effectively blockfibrosis without affecting wound healing. These studies provide thepotential for GSKJ4 treatment to be applied at the time of surgery toblock fibrosis from occurring without the preventing the normal woundhealing process.

FIG. 15 presents the general model of the events that were uncovered onnascent DNA following cataract surgery. Prior to surgery, nascentchromatin is decondensed as evidenced by slow accumulation of H3K27me3mark on DNA. This state of decondensed chromatin is maintained by theactivity of the H3K27me3 KDM UTX that is induced in the region ofsurgery (ECZ). This open chromatin structure allows MRTF-A to bind toits sites on DNA and trigger changes in the transcriptional programleading to differentiation of progenitor cells into the myofibroblasts,hence fibrotic scarring.

FIG. 16 is a model depicting how using epigenetic inhibitors toUTX/JMJD3 can manipulate chromatin structure to block reprogramming to amyofibroblast phenotype. During myofibroblast differentiationdemethylation of H3K27me3 by UTX/JMJD3 on DNA can lead to open chromatinfor the transcription factor binding to induce myofibroblastdifferentiation. In contrast, inhibiting UTX/JMJD3 function by GSKJ4 canlead to closed chromatin structure preventing TF binding, such asMRTF-A, and blocking myofibroblast differentiation.

Therefore, the epigenetic-based therapeutic approach we describe iseffective in preventing fibrosis in situations in which we can access aninjury or dysmorphogenic tissue before the fibrotic process becomesestablished in the tissue. Good examples where such treatment would beespecially effective are injury/surgery/disease in the visual systemincluding the lens post-cataract surgery fibrotic disease PosteriorCapsule Opacification (PCO), hazing/opacification of the corneafollowing cornea injury/surgery including Lasik, age-related maculardegeneration (AMD), scarring following surgery of all types, scarringfollowing skin injury.

Accordingly, to prevent formation of fibrosis we can: condense thechromatin, prevent reprogramming of the myofibroblasts to a fibroticphenotype, and blocking enzymatic activity of the H3K27 de-methylaseUTX. Preferably, one or more actions can be prevented throughadministration of a therapeutic molecule to a patient in need thereof.

Broadly, therapeutic compounds include those sufficient to inhibitJMJD3/UTX. A class of compounds sufficient to inhibit JMJD3 includesthose cited in WO 2012/052390. Those of ordinary skill in the art willrecognize that these molecules can be formulated into appropriatetherapeutic treatments, including those suitable for injection, oralingestion, inhalation, topical administration and the like. However, themost suitable compound is GSK-J4.

As used herein, the term “effective amount” means that amount of a drugor pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system, animal or human that is being sought, forinstance, by a researcher or clinician. Furthermore, the term“therapeutically effective amount” means any amount which, as comparedto a corresponding subject who has not received such amount, results inimproved treatment, healing, prevention, or amelioration of a disease,disorder, or side effect, or a decrease in the rate of advancement of adisease or disorder. The term also includes within its scope amountseffective to enhance normal physiological function.

The compounds suitable as a JMJD3/UTX inhibitor may exist in solid orliquid form. In solid form, compound of the invention may exist in acontinuum of solid states ranging from fully amorphous to fullycrystalline. The term ‘amorphous’ refers to a state in which thematerial lacks long range order at the molecular level and, dependingupon the temperature, may exhibit the physical properties of a solid ora liquid. Typically such materials do not give distinctive X-raydiffraction patterns and, while exhibiting the properties of a solid,are more formally described as a liquid. Upon heating, a change fromsolid to liquid properties occurs which is characterized by a change ofstate, typically second order (‘glass transition’). The term‘crystalline’ refers to a solid phase in which the material has aregular ordered internal structure at the molecular level and gives adistinctive X-ray diffraction pattern with defined peaks. Such materialswhen heated sufficiently will also exhibit the properties of a liquid,but the change from solid to liquid is characterized by a phase change,typically first order (‘melting point’).

The compound suitable as a JMJD3/UTX inhibitor may exist in solvated andunsolvated forms. As used herein, the term “solvate” refers to a complexof variable stoichiometry formed by a solute (in this invention, acompound suitable as a JMJD3/UTX inhibitor or a salt) and a solvent.Such solvents for the purpose of the invention may not interfere withthe biological activity of the solute. The skilled artisan willappreciate that pharmaceutically acceptable solvates may be formed forcrystalline compounds wherein solvent molecules are incorporated intothe crystalline lattice during crystallization. The incorporated solventmolecules may be water molecules or non-aqueous such as ethanol,isopropanol, DMSO, acetic acid, ethanolamine, and ethyl acetatemolecules. Crystalline lattice incorporated with water molecules aretypically referred to as “hydrates”. Hydrates include stoichiometrichydrates as well as compositions containing variable amounts of water.The present invention includes all such solvates. The compounds suitableas a JMJD3/UTX inhibitor may have the ability to crystallize in morethan one form, a characteristic, which is known as polymorphism, and itis understood that such polymorphic forms (“polymorphs”) are within thescope of the invention. Polymorphism generally can occur as a responseto changes in temperature or pressure or both and can also result fromvariations in the crystallization process. Polymorphs can bedistinguished by various physical characteristics known in the art suchas x-ray diffraction patterns, solubility and melting point.

Pharmaceutical compositions may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose.Preferred unit dosage compositions are those containing a daily dose orsub-dose, or an appropriate fraction thereof, of an active ingredient.Such unit doses may therefore be administered once or more than once aday. Such pharmaceutical compositions may be prepared by any of themethods well known in the pharmacy art. Pharmaceutical compositions maybe adapted for administration by any appropriate route, for example bythe oral (including buccal or sublingual), rectal, inhaled, intranasal,topical (including buccal, sublingual or transdermal), vaginal orparenteral (including subcutaneous, intramuscular, intravenous orintradermal) route. Such compositions may be prepared by any methodknown in the art of pharmacy, for example by bringing into associationthe active ingredient with the carrier(s) or excipient(s).

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions. For instance, for oral administration in the form of atablet or capsule, the active drug component can be combined with anoral, non-toxic pharmaceutically acceptable inert carrier such asethanol, glycerol, water and the like. Powders are prepared by reducingthe compound to a suitable fine size and mixing with a similarlyprepared pharmaceutical carrier such as an edible carbohydrate, as, forexample, starch or mannitol. Flavouring, preservative, dispersing andcolouring agent can also be present.

Capsules are made by preparing a powder mixture, as described above, andfilling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilizing agent such asagar-agar, calcium carbonate or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.Moreover, when desired or necessary, suitable binders, glidants,lubricants, sweetening agents, flavours, disintegrating agents andcolouring agents can also be incorporated into the mixture. Suitablebinders include starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like. Disintegratorsinclude, without limitation, starch, methyl cellulose, agar, bentonite,xanthan gum and the like. Tablets are formulated, for example, bypreparing a powder mixture, granulating or slugging, adding a lubricantand disintegrant and pressing into tablets. A powder mixture is preparedby mixing the compound, suitably comminuted, with a diluent or base asdescribed above, and optionally, with a binder such ascarboxymethylcellulose, an alginate, gelatin, or polyvinyl pyrrolidone,a solution retardant such as paraffin, a resorption accelerator such asa quaternary salt and/or an absorption agent such as bentonite, kaolinor dicalcium phosphate. The powder mixture can be granulated by wettingwith a binder such as syrup, starch paste, acadia mucilage or solutionsof cellulosic or polymeric materials and forcing through a screen. As analternative to granulating, the powder mixture can be run through thetablet machine and the result is imperfectly formed slugs broken intogranules. The granules can be lubricated to prevent sticking to thetablet forming dies by means of the addition of stearic acid, a stearatesalt, talc or mineral oil. The lubricated mixture is then compressedinto tablets. The compounds of the present invention can also becombined with a free flowing inert carrier and compressed into tabletsdirectly without going through the granulating or slugging steps. Aclear or opaque protective coating consisting of a sealing coat ofshellac, a coating of sugar or polymeric material and a polish coatingof wax can be provided. Dyestuffs can be added to these coatings todistinguish different unit dosages. Oral fluids such as solution, syrupsand elixirs can be prepared in dosage unit form so that a given quantitycontains a predetermined amount of the compound. Syrups can be preparedby dissolving the compound in a suitably flavoured aqueous solution,while elixirs are prepared through the use of a non-toxic alcoholicvehicle. Suspensions can be formulated by dispersing the compound in anon-toxic vehicle. Solubilizers and emulsifiers such as ethoxylatedisostearyl alcohols and polyoxy ethylene sorbitol ethers, preservatives,flavor additive such as peppermint oil or natural sweeteners orsaccharin or other artificial sweeteners, and the like can also beadded.

Where appropriate, dosage unit compositions for oral administration canbe microencapsulated. The composition can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

The compounds suitable as a JMJD3/UTX inhibitor or a pharmaceuticallyacceptable salt thereof may also be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils. For treatments of theeye or other external tissues, for example mouth and skin, thecompositions are preferably applied as a topical ointment or cream. Whenformulated in an ointment, the active ingredient may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredient may be formulated in a cream with an oil-in-watercream base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administrations to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for rectal administration may bepresented as suppositories or as enemas. Dosage forms for nasal orinhaled administration may conveniently be formulated as aerosols,solutions, suspensions drops, gels or dry powders.

For compositions suitable and/or adapted for inhaled administration, itis preferred that the agent is in a particle-size-reduced form, and morepreferably the size-reduced form is obtained or obtainable bymicronization. The preferable particle size of the size-reduced (e.g.micronized) compound or salt or solvate is defined by a D50 value ofabout 0.5 to about 10 microns (for example as measured using laserdiffraction). Compositions adapted for administration by inhalationinclude the particle dusts or mists. Suitable compositions wherein thecarrier is a liquid for administration as a nasal spray or drops includeaqueous or oil solutions/suspensions of the active ingredient which maybe generated by means of various types of metered dose pressurizedaerosols, nebulizers or insufflators.

Aerosol formulations, e.g. for inhaled administration, can comprise asolution or fine suspension of the agent in a pharmaceuticallyacceptable aqueous or non-aqueous solvent. Aerosol formulations can bepresented in single or multidose quantities in sterile form in a sealedcontainer, which can take the form of a cartridge or refill for use withan atomising device or inhaler. Alternatively the sealed container maybe a unitary dispensing device such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve (metered dose inhaler)which is intended for disposal once the contents of the container havebeen exhausted.

Where the dosage form comprises an aerosol dispenser, it preferablycontains a suitable propellant under pressure such as compressed air,carbon dioxide or an organic propellant such as a hydrofluorocarbon(HFC). Suitable HFC propellants include 1,1,1,2,3,3,3-heptafluoropropane and 1,1,1,2-tetrafluoroethane. The aerosol dosageforms can also take the form of a pump-atomiser. The pressurized aerosolmay contain a solution or a suspension of the active compound. This mayrequire the incorporation of additional excipients e.g. co-solventsand/or surfactants to improve the dispersion characteristics andhomogeneity of suspension formulations. Solution formulations may alsorequire the addition of co-solvents such as ethanol. Other excipientmodifiers may also be incorporated to improve, for example, thestability and/or taste and/or fine particle mass characteristics (amountand/or profile) of the formulation.

For pharmaceutical compositions suitable and/or adapted for inhaledadministration, the pharmaceutical composition may be a dry powderinhalable composition. Such a composition can comprise a powder basesuch as lactose, glucose, trehalose, mannitol or starch, the agent,(preferably in particle-size-reduced form, e.g. in micronized form), andoptionally a performance modifier such as L-leucine or another aminoacid, cellobiose octaacetate and/or metals salts of stearic acid such asmagnesium or calcium stearate. Aerosol formulations are preferablyarranged so that each metered dose or “puff of aerosol contains aparticular amount of a compound of the invention. Administration may beonce daily or several times daily, for example 2, 3 4 or 8 times, givingfor example 1, 2 or 3 doses each time. The overall daily dose and themetered dose delivered by capsules and cartridges in an inhaler orinsufflator will generally be double those with aerosol formulations.

Pharmaceutical compositions adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations. Pharmaceutical compositions adapted for parentaladministration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteriostats andsolutes which render the composition isotonic with the blood of theintended recipient; and aqueous and nonaqueous sterile suspensions whichmay include suspending agents and thickening agents. The compositionsmay be presented in unit-dose or multi-dose containers, for examplesealed ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets.

It should be understood that in addition to the ingredients particularlymentioned above, the compositions may include other agents conventionalin the art having regard to the type of formulation in question, forexample those suitable for oral administration may include flavouringagents.

To further review compounds in this class, we evaluated GSKJ1, GSKJ2,GSKJ4, and GSKJ5. Based on our data, and review of the molecule, thehighly polar carboxylate group of GSKJ1 and GSKJ2 restricts cellularpermeability. This poses potential problems for therapeuticadministration, and indeed, our data shows that by masking the polarityof the acid groups of both the GSKJ1 and GSKJ2 with an ethyl ester, weyield GSKJ4 and GSKJ5 and greater efficacy. GSKJ4 increasedbioavailability, while GSKJ5 was inactive. In in vitro studies, thoughGSKJ1 and GSKJ2 remain potent over a control (data not shown).Accordingly, for purposes of treating disease and disorders here, themost preferred active pharmaceutical compound is GSK-J4.

The formula for GSK-J4 is:

Accordingly, a method of preventing or reducing the formation offibrosis comprises administering to a patient in need thereof, aneffective amount of a histone H3K27 trimethylase (KDM) UTX inhibitor.Preferably, the inhibitor is GSK-J4.

We can also prevent the emergence of αSMA+ myofibroblasts by inhibitingUTX activity through administration of an effective amount of a UTXinhibitor, preferably GSKJ4.

We can also prevent the formation of excessive extracellular matrixproduction in many cases by administering to the patient an effectiveamount of an UTX inhibitor GSK-J4. For example, a preferred method oftreatment includes where a medical practitioner must cut or penetratethe skin of a patient, such as in a surgical procedure, andadministering to the patient having said surgical procedure, aneffective amount of an UTX inhibitor, suitable to increase the number ofH3K27me3 and thereby reducing the formation of excessive extracellularmatrix.

A method of reducing the formation of fibrosis in a patient comprisingblocking the enzymatic activities of UTX and JMJD3 by administering tosaid patient an effective amount of a UTX and JMJD3 inhibitor GSKJ4.

A method of preventing the onset and progression of a fibrotic diseaseby blocking pro-fibrotic transcription factors from binding tochromatin, comprising administering an effective amount of a therapeuticGSKJ4 sufficient to prevent the binding of pro-fibrotic transcriptionfactor MRTF-A.

A method of treating or preventing the onset of fibrosis after asurgical procedure comprising: Performing a surgical procedure on apatient; administering to said patient an effective amount of a UTX andJMJD3 inhibitor. Preferably, the UTX and JMJD3 inhibitor is administeredwithin 24 hours of the surgical procedure, and more preferably the UTXand JMJD3 inhibitor is administered to the patient within 16, 8, 4, 3,2, 1 hour, or within 30 minutes, 15 minutes, 5 minutes, orsimultaneously with the surgical procedure. Furthermore,re-administration of a further dose, or a sustained release dose isprovided to maintain elevated levels of the inhibitor for a period ofabout 12-72 hours, preferably for about 12-48 hours, and more preferablyfor at least 24 hours.

For example, a patient is undergoing cataract surgery of the eye, itwould be advantageous to perform the surgery and then provide the UTXand JMJD3 inhibitor to the patient during and after the procedure, butnot more than 24 hours after the completion of the procedure. Thisallows for accumulation of H3K27me3, and thus allows the H3K27me3 tobind to the chromatin and generate or maintain a closed chromatinstructure. Preferably, the UTX and JMJD3 inhibitor is GSKJ4, and isapplied topically to the eye during the surgical procedure. A furtherstep for such cataract surgery is the placement of an intra ocular lens,the IOC may be coated with a powder, suspension, or gel, or otherdelivery vehicle, to allow for delivery of the therapeutic to the eyefor a sufficient period to prevent fibrosis formation. Preferably, thisincludes a period of at least the time from surgery to a period of about24 hours after surgery. While additional exposure may be suitable, i.e.treatment for 3-7 days, the short treatment protocol is advantageous.

A preferred embodiment is directed to preventing or reducing theformation of fibrosis after a surgical procedure, comprising performinga surgical procedure, administering to the patient and effective amountof a UTX and JMJD3 inhibitor within 24 hours of the surgical procedure.Preferably, the UTX and JMJD3 inhibitor is provided at the time of thesurgical procedure and is given for about 24 hours. For example, for aprocedure on the eye, a topical administration may be provided at thetime of the procedure and then given at least one additional time withinthe next 24 hour period. A short treatment is surprisingly effectiveover this 24 hour period to reduce and prevent the formation offibrosis.

For example, when administering GSK-J4 to a patient, an appropriatetherapeutic range comprises: about 1 μM to 10 μM concentration for aliquid, topical vehicle. Suitable doses can be generated based upon thetissue to be treated and area to be treated. Testing at the lower end ofthe range revealed that there was higher efficacy at 10 μM than the 1μM, however, a further 10× increase to 100 μM began to show signs oftoxicity. The LD50 is otherwise known to those of skill in the art basedupon the molecule of interest. The GSK-J4 can be formulated withsuitable excipients and into a suitable vehicle for delivery to a humanpatient.

A preferred embodiment comprises administration of a H3K27 de-methylaseUTX inhibitor during and after a surgical procedure for 24 hours,sufficient to reduce or prevent the formation of fibrosis due to saidsurgical procedure. Preferably, the process includes administering tothe patient undergoing the surgical procedure, an effective amount ofsaid UTX inhibitor during the surgical process and at least one furtherdose of said UTX inhibitor within 24 hours of the surgical process. Thebenefit of the short timeframe is that the UTX inhibitor is effective toprevent re-programming of myofibroblast cells within this brief windowis sufficient to prevent the re-programming and also allows for healingof the wound.

Depending on the surgical procedure, it may be suitable to administer acrème, gel, paste, suspension, or emulsion, in a pharmaceuticallysuitable carrier to the site of the surgical procedure. For example, anocular surgery may use a direct application to the eye, the eye ductsand the like, wherein the active pharmaceutical material will reach theeye tissues that are at risk for becoming fibrotic. Similarly, asurgical procedure that requires an incision to the body may utilize asimilar topical material, to prevent or reduce fibrosis formation at theincision site. Furthermore, in each the eye or the incision procedures,the pharmaceutical composition may be administered intradermally,through injection with a hypodermic needle to the skin tissuessurrounding the skin tissues undergoing the surgical procedure.

Indeed, for procedures within the body, for example repaid or a rupturedtendon or major muscle tear, requiring surgical intervention, theinternal tissues may be disposed to fibrosis. Accordingly, intramuscularinjections of a pharmaceutical composition to the wound, injury, orsurgical site will be effective for treating or preventing the formationof fibrosis. Surgeons of ordinary skill will be able to effectivelyadminister the pharmaceutical composition into these wound sites and tochoose the proper delivery vehicle, including injectable, solid, gel,paste, or other compositions formulated for internal delivery to thebody.

Finally, for certain internal applications, it may be more effective foran oral application or inhaled application of the pharmaceuticalcomposition. In such a case, an oral dosage form can be administeredduring a short treatment, typically for the first 24 hours after thesurgical procedure.

Accordingly, the processes and methods described herein provide atherapeutic use of a pharmaceutical composition an inhibitor of UTXand/or JMJD3, preferably GSK-J4. Use of the inhibitor is effective inensuring a condensed structure of chromatin and preventing there-programming of myofibroblasts into a fibrotic cell. Accordingly, thetherapeutic use of the pharmaceutical composition is effective inpreventing or reducing the formation of fibrotic tissues.

What is claimed is:
 1. A method of preventing or reducing fibrosis,comprising administering to a patient an effective amount of apharmaceutical composition comprising a H3K27 de-methylase UTXinhibitor.
 2. The method of claim 1 wherein the H3K27 de-methylase UTXinhibitor is GSK-J4 or a suitable salt thereof.
 3. The method of claim1, wherein the pharmaceutical composition is administered topically to askin surface.
 4. The method of claim 2, wherein the pharmaceuticalcomposition has a concentration of between 1 μM and 10 μM GSK-J4.
 5. Themethod of claim 4, wherein the pharmaceutical composition has aconcentration of 10 μM GSK-J4.
 6. The method of claim 1, wherein thepharmaceutical composition is administered to a patient within 24 hoursof a surgical procedure.
 7. The method of claim 1, wherein thepharmaceutical composition is administered to a patient at the time ofsurgery.
 8. A method of treatment of a patient tissues before thefibrotic process establishes after a surgical procedure, comprisingadministering to said patient an effective amount of a pharmaceuticalcomposition comprising a H3K27 de-methylase UTX inhibitor at the time ofthe surgical procedure.
 9. The method of claim 8, wherein the H3K27de-methylase UTX inhibitor is GSK-J4 or a suitable salt thereof.
 10. Themethod of claim 8, wherein the pharmaceutical composition isadministered orally, intradermally, or topically to a skin surface. 11.A method of preventing or reducing the formation of fibrosis in oculartissues after an ocular surgery comprising: administering to a patientundergoing said ocular surgery a UTX inhibitor at the time of the ocularsurgery.
 12. The method of claim 11, wherein the UTX inhibitor is GSK-J4or a suitable salt thereof.
 13. The method of claim 11, wherein the UTXinhibitor is administered topically to the eye.
 14. A method oftreatment of the fibrotic disease Posterior Capsule Opacification (PCO)comprising: performing a cataract surgery on a patient; andadministering to said patient a sufficient amount of a UTX inhibitorduring said surgery.
 15. The method of claim 14 further comprisingimplanting an intraocular lens into the eye.
 16. The method of claim 15,wherein said intraocular lens comprises a sustained release mechanism toallow for release of the UTX inhibitor for at least 24 hours.
 17. Themethod of claim 16, wherein said sustained release mechanism providesfor release for between 24 and 72 hours.
 18. The method of claim 14,wherein the UTX inhibitor is GSK-J4 or a suitable salt thereof.
 19. Themethod of claim 14, wherein the UTX inhibitor is administered topicallyto the eye.